Inhibitors of factor Xa

ABSTRACT

Novel compounds, their salts and compositions related thereto having activity against mammalian factor Xa are disclosed. The novel compounds include peptide aldehyde analogues having substantial potency and specificity as inhibitors of mammalian factor Xa are further disclosed. The compounds are thought useful as inhibitors of factor Xa in vitro or as a therapeutic agent for the prevention and treatment of conditions: characterized by abnormal thrombosis in mammals. Intermediates useful for the preparation of the novel compounds are also disclosed.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/168,964, filed Dec.15, 1993, which in turn is a continuation-in-part of U.S. Ser. No.07/991,204, filed Dec. 15, 1992, entitled "Novel Inhibitors of FactorXa", now abandoned, the disclosure of which is hereby incorporated byreference including the drawings attached thereto.

FIELD OF THE INVENTION

The present invention relates in one aspect to compounds, theirpharmaceutically acceptable salts and pharmaceutically acceptablecompositions thereof which are potent and specific inhibitors of bloodcoagulation in mammals. The inhibition of clot formation flows from thedirect inhibition of the blood coagulation enzyme, factor xa, by theinhibitors disclosed. In another aspect, the invention relates to amethods of using these inhibitors in their various embodiments astherapeutic agents for disease states characterized by disorders of theblood coagulation process. In yet another aspect, the invention relatesto intermediate compounds useful in the preparation of the inhibitors.

BACKGROUND AND INTRODUCTION TO THE INVENTION

Normal hemostasis is the result of a complex balance between theprocesses of clot formation (blood coagulation) and clot dissolution(fibrinolysis). The complex interactions between blood cells, specificplasma proteins and the vascular surface maintain the fluidity of bloodunless injury and blood loss occur. Many significant disease states arerelated to abnormal hemostasis. Abnormal thrombus formation occurring inthe coronary arterial vasculature due to the rupture of an establishedatherosclerotic plaque is the major cause of acute myocardial infarctionand unstable angina. Treatment of occlusive coronary thrombus by eitherthrombolytic therapy or percutaneous transluminal angioplasty is oftenaccompanied by an acute thrombotic reclosure of the affected vesselwhich requires immediate resolution. A high percentage of patientsundergoing major surgery in the lower extremities or the abdominal areasuffer from thrombus formation in the venous vasculature which canresult in reduced blood flow to the affected extremity and apredisposition to pulmonary embolism. Disseminated intravascularcoagulopathy commonly occurs during septic shock, certain viralinfections and cancer and is characterized by the rapid consumption ofcoagulation factors and systemic coagulation which results in theformation of life-threatening thrombi occurring throughout thevasculature leading to wide-spread organ failure.

Blood coagulation is the culmination of a series of amplified reactionsin which several specific zymogens of serine proteases in plasma areactivated by limited proteolysis. This results in the formation of aninsoluble fibrin matrix which is required for the stabilization of theprimary hemostatic plug. The interaction and propagation of theactivation reactions occurs through the extrinsic and intrinsic pathwaysof coagulation as reviewed by Mackie, I. J. and Bull, H. A, "NormalHemostasis and its Regulation" Blood Reviews, 3:237-250 (1989). Bothpathways are highly inter-dependent and converge in the formation of theserine protease factor Xa from its zymogen, factor X. Factor Xacatalyzes the penultimate step in the blood coagulation cascade which isthe formation of the serine protease thrombin. Thrombin goes on tocleave soluble fibrinogen in the plasma to form insoluble fibrin.

The biochemical and physiological characterization of factor X has beenreviewed by Steinberg, M. and Nemerson, Y, "Activation of Factor X",Hemostasis and Thrombosis, First Edit, pp 91-111 (Colman, R. et. al.eds. 1982) and Mann, K. G. et. al, "Surface-Dependent Reactions of theVitamin K-Dependent Enzyme Complexes", Blood, 76:1-16 (1990). Humanfactor X circulates in plasma at a concentration of 170 nM. The enzymeis a two-chain glycoprotein containing 442 amino acid residues having anoverall molecular size (Mr) of 59,000 as determined by sedimentationequilibrium centrifugation and approximately 67,000 by sodium dodecylsulfate electrophoresis. DiScipio et. al, "A comparison of humanprothrombin, Factor IX (Christmas Factor), Factor X (Stuart Factor) andProtein S", Biochemistry, 16:698-706 (1977) and Leyfus et. al,"Characterization of a cDNA coding for human Factor X", Proc. Natl.Acad. sci. USA, 82:3699 (1984). Human factor X contains a light-chainsubunit containing 139 amino acid residues (Mr=16,200) and a heavy chainsubunit containing 303 amino acid residues (Mr=42,000) linked togetherby a single disulfide bond. The light chain of human factor X contains11 glutamic acid residues which have been post-translationally modifiedto γ-carboxy-glutamic acid and one asparagine acid moiety modified toβ-hydroxy aspartic acid. The heavy chain of factor X contains all of theglycosylated residues (15% overall) as well as the catalytic domain ofthe molecule.

Proteolytic activation of zymogen factor X to its catalytically activeform, factor Xa, can occur by either the intrinsic or extrinsiccoagulation pathways. The intrinsic pathway is referred to as intrinsicbecause everything needed for clotting is in the blood. Saito, H0,"Normal Hemostatic Mechanisms", Disorders of Hemostasis, pp. 27-29,Grune & Stratton, Inc. (O. D. Ratnoff, M. D. and C. D. Forbes, M. D.edit. 1984). This pathway is comprised of the zymogen serine proteases,factors IX and XI, and the non-enzymatic co-factor, factor VIII. Theinitiation of the intrinsic pathway results in the activation of factorXI to XIa. Factor XIa catalyzes the activation of factor IX to factorIXa which in combination with the activated form of factor VIII on anappropriate phospholipid surface, results in the formation of the tenasecomplex. This complex also catalyzes the formation of the serineprotease, factor Xa, from its zymogen, factor X, which subsequentlyresults in clot formation.

The extrinsic pathway is referred to as extrinsic because the tissuefactor which binds to and begins activation of factor VII comes fromoutside the blood. Saito, Id. The major components of this pathway arethe zymogen serine protease, factor VII, and the membrane bound protein,tissue factor. The latter serves as the requisite non-enzymaticco-factor for this enzyme. The initiation of this pathway is thought tobe an autocatalytic event resulting from the activation of zymogenfactor VII by trace levels of activated factor VII (factor VIIa), bothof which are bound to newly exposed tissue factor on membrane surfacesat sites of vascular damage. The factor VIIa/tissue factor complexdirectly catalyzes the formation of the serine protease, factor Xa, fromits zymogen, factor X. Exposure of blood to injured tissue initiatesblood clotting by the extrinsic pathway.

Proteolytic activation of zymogen factor X to its catalytically activeform, factor Xa, results in the liberation of a 52 amino acid activationpeptide from the amino-terminus of the heavy chain subunit. Theintrinsic activation reaction is catalyzed by factor iXa in amacromolecular complex with the non-enzymatic co-factor, factor VIII.Factor Xa formation via the extrinsic pathway is catalyzed by thecatalytic complex of factor VIIa and tissue factor. Both of thesereactions must occur on an appropriate phospholipid surface in thepresence of calcium ions. The active product formed following eitherintrinsic or extrinsic activation of factor X is α-factor Xa. A secondproteolytic cleavage which is thought to be autocatalytic, results inthe formation of β-factor Xa following the release of a 14 amino acidpeptide from the carboxy-terminus of the heavy chain. Both forms of theactivated molecule have the same catalytic activity as measured by theirability to promote coagulation in plasma or hydrolyze a peptidylchromogenic substrate.

The formation of thrombin is catalyzed by factor Xa following theassembly of the catalytic prothrombinase complex as reviewed by Mann, K.G. et. al, "Surface-Dependent Reactions of the Vitamin K-DependentEnzyme Complexes", Blood, 76:1-16 (1990). This complex is composed offactor Xa, the non-enzymatic co-factor Va and the substrate prothrombinall assembled on an appropriate phospholipid surface. The requirement ofa macromolecular complex for efficient catalysis results in theprotection of factor Xa from natural anticoagulant mechanisms such asheparin-antithrombin III mediated inhibition. Teite, J. M. andRosenberg, R. D., "Protection of Factor Xa from neutralization by theheparin-antithrombin complex", J. Clin. invest, 71:1383-1391(1983). Inaddition sequestration of factor Xa in the prothrombinase complex alsorenders it resistant to inhibition by exogenous heparin therapy whichalso requires antithrombin III to elicit its anticoagulant effect.

Several examples of naturally occurring polypeptide inhibitors of factorXa have been reported to have excellent specificity and potency. U.S.Pat. No. 4,588,587 to Gasic describes the anticoagulant activity ofHaementeria offcinalis leech saliva. A principal component of the leechsaliva, Antistasin was said to inhibit factor Xa. See, Tuszynski, G. P.et. al, "Isolation and characterization of antistasin, an inhibitor ofmetastasis and coagulation", J. Biol. Chem, 262:9718-9723 (1987); Nutt,E. et. al, "The amino acid sequence of antistasin, a potent inhibitor ofFactor Xa reveals a repeated internal structure", J. Biol. Chem,63:10162-10167 (1988); Dunwiddie, C. et. al, "Antistasin, aleech-derived inhibitor of factor Xa, kinetic analysis of enzymeinhibition and identification of the reactive site", J. Biol. Chem,264:16694-16699 (1989); and Han, J. H. et. al, "Cloning and expressionof cDNA encoding antistasin, a leech-derived protein havinganti-coagulant and anti-metastatic properties", Gene, 72:47-57 (1989).

A polypeptide reported to be a selective and potent inhibitor of factorXa was originally isolated from whole body extracts of the soft tickOrnithidoros moubata. See Waxman, L. et. al, "Tick anticoagulant peptide(TAP) is a novel inhibitor of blood coagulation factor Xa", Science,248:593-596 (1990); Neeper M. P. et al., "Characterization ofrecombinant tick anticoagulant peptide, a highly selective inhibitor ofblood coagulation factor Xa", J. Biol. Chem, 265:17746-17752 (1990); andJordan, S. P. et. al, "Tick anticoagulant peptide: kinetic analysis ofthe recombinant inhibitor with blood coagulation factor Xa",Biochemistry, 29:11095-11100 (1990); and Vlasuk et al, U.S. Pat. No.5,239,058 Aug. 24, 1993).

Plasma has been reported to contain a common inhibitor of both factor Xaand factor VIIa-tissue factor complex called lipoprotein-associatedcoagulation inhibitor (LACI). LACI is reported to consist of 276 aminoacids and has been reported to inhibit the proteolytic activity offactor Xa directly, and in a factor Xa-dependent manner, factorVIIa-tissue factor complex. Girard, T. J. et al, Nature, 338:518-520(1989).

Other polypeptide inhibitors of factor Xa have also been reported. See,e.g., Jacobs, J. w. et. al, "Isolation and characterization of acoagulation factor Xa inhibitor from Black fly salivary glands", Thromb.Haemostas, 64:235-238 (1990); Condra, C. et. al, "isolation andstructural characterization of a potent inhibitor of coagulation factorXa from the leech Haementeria ghilianii", Thromb. Haemost, 61:437-441(1989); Brankamp, R. G. et. al, "Ghilantens: anticoagulants,antimetastatic proteins from the South American leech Haementeriaghilianii", J. Lab. Clin. Med., 115:89-97 (1990); Blankenship, D. T. et.al, "Amino acid sequence of ghilanten: anti-coagulant-antimetastaticprinciple of the South American leech, Haementeria ghilianii"", Biochem.Biophys. Res. Commun, 166:1384-1389 (1990); and Rigbi, M. et. al.,"Bovine factor Xa inhibiting factor and pharmaceutical compositionscontaining the same", European Patent Application, publication no.352,903 (1990).

In addition to the above polypeptide inhibitors of factor Xa, smallmolecule inhibitors of this enzyme have been reported. See, Kam, et.al., "Mechanism based isocoumarin inhibitors for trypsin and bloodcoagulation serine proteases: new anticoagulants", Biochemistry,27:2547-2557 (1988); Tidwell, R. R. et. al., "Strategies foranticoagulation with synthetic protease inhibitors. Xa inhibitors versusthrombin inhibitors", Thromb. Res, 19:339-349 (1980); Hitomi, Y. st.al., "Inhibitory effect of a new synthetic protease inhibitor (FUT-175)on the coagulation system", Haemostasis, 15:164-168 (1985); furner, A.D. et. al., "p-Amidino esters as irreversible inhibitors of factors IXaand Xa and thrombin", Biochemistry, 25:4929-4935 (1986); andSturzebecher, J. et. al., "Synthetic inhibitors of bovine factor Xa andthrombin. Comparison of their anticoagulant efficiency", Thromb. Res,54:245-252 (1989).

Unlike the reported polypeptide inhibitors of factor xa, the known smallmolecule inhibitors have been reported to be relatively non-selectivewith respect to the inhibition of other serine proteases. For example,6-amidino-2-naphthyl-p-guanidinobenzoate dimethanesulfonate (FUT-75)inhibits human factor Xa and human thrombin similarly, yieldinginhibitor constants of 4.1 μM and 1.3 μM, respectively. Hitomi et al.,supra, at p. 166. p-Amidinophenyl α-methylcinnamate irreversiblyinactivates human factor Xa, human factor XIa and human thrombinsimilarly yielding second-order rate constants of inactivation of9.9×10⁴, 16×10⁴, and 16×10⁴ M⁻¹ min⁻¹, respectively. Turner et al.,supra, at p. 4932.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to compounds whichselectively inhibit the catalytic activity of factor Xa but do notappreciably inhibit the activity of factor XIa, thrombin or tissueplasminogen activator (tPA). These compounds are characterized by havinga Percent Selectivity for each of factor XIa, thrombin and tPA less thanor equal to 10. Percent Selectivity is defined as 100 times the IC₅₀ forfactor Xa divided by the IC₅₀ of either factor XIa, thrombin or tPA.IC₅₀ is that concentration of test compound giving 50% inhibition of thesubstrate turnover. These compounds are peptide analogs of relativelylow molecular weight, namely, peptide aldehydes which have molecularweights less than about 1000. These compounds are thought to be usefuleither as in vitro diagnostic reagents for selectively inhibiting factorXa while only weakly inhibiting, if at all, factor Xia, thrombin ortissue plasminogen activator (tPA), or as pharmacological agents for thetreatment of certain thrombotic disorders.

The novel compounds of the present invention include those representedby the formulas: ##STR1## wherein R₁ is selected from the groupconsisting of --(CH₂)₃ --NH--C(═NH)--NH₂ and mono-anddi-alkyl-substituted derivatives thereof, wherein each alkyl group isindependently selected and has about 1 to about 7 carbon atoms;

R₂ is selected from the group consisting of aralkyl of about 6 to about15 carbon atoms optionally substituted with 1 to 2 independentlyselected alkyl groups of about 1 to about 4 carbon atoms;

R₃ is selected from the group consisting of aryl of about 6 to about 14carbon atoms optionally substituted with 1 to 2 independently selectedalkyl groups of about 1 to about 4 carbon atoms, aralkyl of about 7 toabout 15 carbon atoms optionally substituted with 1 to 2 independentlyselected alkyl groups of about 1 to about 4 carbon atoms, and alkyl ofabout 1 to about 7 carbon atoms; and

R₄ is selected from the group consisting of alkyl of about to about 12carbon atoms, alkenyl of about 3 to about 6 carbon atoms, aryl of about6 to about 14 carbon atoms, aralkyl of about 6 to about 15 carbon atoms,alkoxy of about 1 to about 12 carbon atoms, alkenyloxy of about 3 toabout 8 carbon atoms, aryloxy of about 6 to about 14 carbon atoms,araloxy of about 6 to about 15 carbon atoms, and carboxyalkyl of about 2to about 7 carbon atoms.

The present invention also encompasses the pharmaceutically acceptablesalts of the compounds of formulas (I) and (I'). These salts includeacid addition salts, for example, salts of hydrochloric acid,hydrobromic acid, acetic acid, benzene sulfonic acid and other suitableacid addition salts, including buffering salts.

Peptidyl arginine aldehydes have been reported to exist in equilibriumstructures in aqueous solutions. Bajusz, S, et al., J. Med Chem.,33:1729 (1990). These structures, as shown below, include the argininealdehyde, A, aldehyde hydrate, B, and two amino cyclol forms, C and D.The R group would represent the remainder of a given compound embodiedin the present invention. The peptide aldehydes of the present inventioninclude within their definition all its equilibrium forms. ##STR2##

In another aspect, the present invention is directed to compounds whichare intermediates for the novel compounds claimed herein. Theintermediates include those represented by the formulas: ##STR3##wherein Ar has the formula: ##STR4## where each X is independentlyselected from the group consisting of hydrogen, methyl, methoxy,halogen, ethyl and ethoxy; R₁ is selected from a group consisting of--(CH₂)₃ --NH--C(═NNO₂)--NH₂, and mono- or di-alkyl-substitutedderivatives thereof, wherein each alkyl group is independently selectedand has about 1 to about 7 carbon atoms; and R₂, R₃ and R₄ are asdefined as for formulas (I) and (I') hereinabove.

The present invention also provides compositions and methods forpreventing or treating a condition characterized by abnormal thrombusformation in mammals.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups.

The term "alkenyl" refers to unsaturated hydrocarbyl groups whichcontain at least one carbon-carbon double bond and includesstraight-chain, branched-chain and cyclic groups.

The term "aryl" refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,and may be optionally substituted.

The term "methylene" refers to --CH₂ --.

The term "alkylene" refers to a divalent straight chain or branchedchain saturated aliphatic radical

The term "alkoxy" refers to the group --OR, wherein R is alkyl.

The term "alkenyloxy" refers to the group --OR, wherein R is alkenyl.

The term "aryloxy" refers to the group --OR, wherein R is aryl.

The term "aralkyloxy" refers to the group --OR, wherein R is aralkyl.

The term "carboxyalkyl" refers to the group --alk--COOH, wherein alk isan alkylene group.

The term "halogen" refers to fluorine, chlorine, bromine or iodineatoms.

The term "pharmaceutically acceptable salt" includes salts of thecompounds of the present invention derived from the combination of asuch compounds and an organic or inorganic acid. In practice, the use ofthe salt form amounts to use of the base form. The compounds of thepresent invention are useful in both free base and salt form, with bothforms being considered as being witlhin the scope of the presentinvention.

In addition, the following abbreviations stand for the following:

"Ac" refers to acetyl.

"α-NpAla" refers to 3-(1-naphthyl )alanine also known as3-(α-naphthyl)alanine.

"β-NpAla" refers to 3-(2-naphthyl)alanine also known as3-(β-naphthyl)alanine.

"Bn" refers to benzyl.

"Boc" refers to t-butoxycarbonyl .

"BOP" refers tobenzotriazol-1-yloxy-tris--(dimethylamino)-phosphonium-hexafluorophosphate.

"BPGly" refers to α-biphenylglycine.

"Brine" means an aqueous saturated solution of sodium chloride.

"CDI" refers to carbonyldiimidazole.

"DCM" refers to dichloromethane.

"DIEA" refers to diisopropylethylamine.

"DMF" refers to N,N-dimethylformamide.

"IPA" refers to isopropanol.

"MeOH" refers to methanol.

"4MeV" refers to 4-methylvaleroyl.

"NaOAc" refers to sodium acetate.

"NMM" refers to 4-methylmorpholine.

"Ph" refers to phenyl group.

"PhGly" refers to 2-phenylglycine.

"Ppa" refers to a protected peptide analog.

"Succ" refers to succinyl.

"TBSA" refers to 0.1M Tris, 0.14M sodium chloride, pH 7.4 containing0.1% bovine serum albumin.

"TEA" refers to triethylamine.

"TFA" refers to trifluoroacetic acid.

"THF" refers to tetrahydrofuran.

"3-trans-PhPro" refers to 3-trans-phenyl-L-proline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a reaction scheme describing a process for preparing asolid-phase reagent, 1, which may be subsequently used to make one ormore of the compounds of the present invention. In this figure, Bnrepresents to benzyl; t-Bu represents to t-butyl; and Boc represents tot-butoxycarbonyl.

FIG. 2 depicts a reaction scheme describing a process for synthesis ofcompound which may subsequently be used to make one or more of thecompounds of the present invention. In this figure, i representsp-toulenesulfonic acid and benzyl alcohol; ii representsBoc-D-phenylalanine, BOP and NMM in DMF; and iii represents hydrogen gasat 30 psig with 10% palladium on carbon in THF.

FIG. 3 depicts a reaction scheme describing a process for synthesis ofone compound of the present invention using a liquid-phase method. Inthis figure, i represents t-butylcarbazate and carbonyldiimidazole inDMF under nitrogen; ii represents trifluoroacetic acid at 0° C.; iiirepresents compound 1 with sodium acetate in refluxing ethanol/water; ivrepresents trifluoroacetic acid in dichloromethane at 0° C.; vrepresents a protected peptide analog ("Ppa") such as compound 22! withBOP and NMM in DMF; vi represents hydrogen gas at 15 psig with 10%palladium on carbon in acidified methanol/acetic acid or HF/anisolefollowed by vii; and vii represents formaldehyde in acidifiedmethanol/acetic acid.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention have been found to have thesurprising property that they are potent inhibitors of factor Xa butonly weak inhibitors of factor XIa, thrombin and tissue plasiminogenactivator (tPA). Significantly, previously reported small-moleculefactor Xa inhibitors which have been described as effectiveantithrombotic agents are nonspecific in their inhibitory activity inthat they cannot distinguish between the factor Xa and other coagulationenzymes. Also, peptide aldehyde derivatives have been described asineffective in inhibiting factor Xa and this inability has beendescribed as a limitation of the use of peptide aldehyde inhibitors inblood coagulation. Bajusz, S. etal, "Design and Synthesis of Peptideinhibitors of Blood Coagulation", Folia Haematol, Leipzig, 109:16 at 19(1982).

Preferred Compounds

One aspect of the present invention is directed to N-acyl derivatives ofcertain peptide aldehydes. These compounds are characterized by theirability to strongly inhibit factor Xa but only weakly inhibit factorXIa, thrombin and tPA. Formulas (I) and (I') below depict the compoundscomprising this aspect of the present invention: ##STR5## wherein R₁ isselected from the group consisting of --(CH₂)₃ --NH--C(═NH)--NH₂ andmono- or di-alkyl-substituted derivatives thereof, wherein each alkylgroup is independently selected and has about 1 to about 7 carbon atoms;

R₂ is selected from the group consisting of aralkyl of about 6 to about15 carbon atoms optionally substituted with 1 to 2 independentlyselected alkyl groups of about 1 to about 4 carbon;

R₃ is selected from the group consisting of aryl of about 6 to about 14carbon atoms optionally substituted with 1 to 2 independently selectedalkyl groups of about 1 to about 4 carbon atoms, aralkyl of about 7 toabout 15 carbon atoms optionally substituted with 1 to 2 independentlyselected alkyl groups of about 1 to about 4 carbon atoms, and alkyl ofabout 1 to about 7 carbon atoms; and

R₄ is selected from the group consisting of alkyl of about 1 to about 12carbon atoms, alkenyl of about 3 to about 6 carbon atoms, aryl of about6 to about 15 carbon atoms, aralkyl of about 6 to about 15 carbon atoms,alkoxy of about 1 to about 12 carbon atoms, alkenyloxy of about 3 toabout 8 carbon atoms, aryloxy of about 6 to about 14 carbon atoms,aralkyloxy of about 6 to about 15 carbon atoms and carboxyalkyl of about2 to about 7 carbon atoms.

The present invention also encompasses the pharmaceutically acceptablesalts of these compounds. These salts include acid addition salts, forexample, salts of hydrochloric acid, hydrobromic acid, acetic acid,benzene sulfonic acid and other suitable acid addition salts, includingbuffering salts.

For convenience in discussing preferred substituents, these compoundscan be divided into parts as shown in the following formulas (Ia) and(Ia'): ##STR6## Preferred compounds include those where the P1 aminoacid analogue is an L-isomer. Especially preferred are the compoundswherein R₁ is --(CH₂)₃ --NH--C(═NH)--NH₂.

These preferred compounds of formula (I) and (Ia) also contain a P₂amino acid analogue which is a L-isomer, containing an R₂ group which ispreferably an aralkyl such as phenylmethyl, diphenylmethyl, biphenylmethyl, naphthylmethyl, or mono- or di-substituted alkyl derivativesthereof, wherein each alkyl group has about 1 to about 4 carbon atoms.Especially preferred are compounds wherein is R₂ is phenylmethyl,1-naphthylmethyl or 2-naphthylmethyl.

The preferred compounds of formula (I) and (Ia) or (I') and (Ia') alsocontain a P₃ amino acid analogue which is a D-isomer, containing an R₃group which may be an aryl or aralkyl group suitable R₃ groups includegroups, such as phenyl, phenylmethyl, diphenylmethyl, biphenyl,biphenylmethyl, napthyl, naphthylmethyl or mono- or di-substituted alkylderivatives thereof, wherein each alkyl group has about 1 to about 4carbon atoms or alkyl groups such as cyclohexylmethyl. Especiallypreferred are compounds wherein R₃ is phenyl, phenylmethyl,1-naphthylmethyl or 2-naphthylmethyl .

The preferred compounds of formula (I) and (Ia) or (I') and (Ia') willalso include a N-acyl group (R₄ --C(O)--), at the N-terminus of thethird amino acid analogue. Suitable R₄ groups include methyl, ethyl,1,1-dimethylethyl, propyl, 2-methylpropyl, 2,2-dimethylpropyl, butyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl , adamantylmethyl, 2-propenyl, 3-butenyl,1-pentenyl, 2-pentenyl, 5-hexenyl, 2-cyclopentenyl, phenyl, phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl, naphthyl, naphthylmethyl,1,1-dimethylethyloxy, 2-methylpropyloxy, 2,2-dimethylpropyloxy,cyclopentyloxy, cyclopentylmethyloxy, cyclohexyloxy,cyclohexylmethyloxy, adamantyloxy, adamantylmethoxy, phenoxy, benzyloxy,biphenylmethyloxy, naphthloxy, naphthylmethyloxy, or 2-carboxyethyl.Especially preferred are compounds wherein R₄ is methyl or1,1-dimethylethyloxy.

Preferred peptide aldehydes of the present invention include: ##STR7##

Another aspect of the present invention is directed to intermediatesuseful for the preparation of the novel N-acyl peptide aldehydederivatives of formulas (I) and (I'). These intermediates are depictedby the formulas (II) and (II') below: ##STR8## wherein R₁, R₂, R₃ and R₄are as defined in connection with formulas (I) and (I') and Ar is anaryl group.

Preferred intermediates include those wherein Ar has the formula:##STR9## where each X is independently selected from the groupconsisting of hydrogen, methyl, halogen, and ethyl. Especially preferredare intermediates where Ar is phenyl.

Preferred intermediates of formulas (II) and (II') include compoundswherein R₁ is --(CH₂)₃ --NH--C(═NNO₂)--NH₂.

Preferred groups for R₂, R₃ and R₄ are the same as those given forformulas (I) and (I') hereinabove. Especially preferred intermediatesinclude those where R₂ is phenylmethyl, 1-naphthylmethyl or2-naphthylmethyl; R₃ is phenyl, phenylmethyl, 1-naphthylmethyl or2-naphthylmethyl; and R₄ is methyl or 1,1-dimethylethyloxy.

Particularly preferred intermediates of the present invention include:##STR10##

Preparation of Preferred Compounds

The peptide aldehyde derivatives of the present invention may besynthesized by either solid or liquid phase methods. Under certainconditions, such as large scale syntheses, the liquid phase methoddescribed herein is preferred.

Starting materials used in the preparation of these compounds by eithermethod are readily available from commercial sources as Aldrich, BachemBioscience Inc, Nova Biochemicals, Sigma and the like.

During the synthesis of these compounds, the functional groups of theamino acid derivatives used in these methods are protected by blockinggroups to prevent cross reaction during the coupling procedure. Examplesof suitable blocking groups and their use in peptide synthesis aredescribed in "The Peptides: Analysis, Synthesis, Biology", AcademicPress, Vol. 3 (E. Gross & Meienhofer edit. 1981) and Vol. 9 (S.Udenfriend & J. Meienhofer edit. 1987), the disclosures of which areincorporated herein by reference.

The peptide aldehyde derivatives of the present invention may besynthesized by procedures described in the literature (see below) or bysequential chemical attachment of amino acid derivatives using the solidphase synthesis reagents and methods described in commonly assigned U.S.Pat. No. 5,367,072, the disclosure of which is incorporated herein byreference.

FIG. 1 herein illustrates the synthesis of a solid phase reagent towhich amino acid derivatives may be later attached in the solid phasesynthesis method.

The peptide aldehyde derivatives of the present invention may also besynthesized by solution phase methods. Preferred is the method depictedin FIGS. 2 and 3. FIG. 2 depicts a process for the synthesis of acompound subsequently used to prepare the compounds of the presentinvention. FIG. 3 depicts a preferred process for the solution phasesynthesis of the compounds of the present invention. Other methods forthe solution synthesis of peptide aldehydes have been reported and maybe used to prepare the compounds of formulas (I) and (I'). For example,see McConnell et al., J. Med. Chem, 33:86, at 87 (1989) and referencescited therein; Kawamura et al., Chem. Pharm. Bull, 17:1902 (1969), andSomeno et al., Chem. Pharm. Bull, 34:1748 (1986).

The intermediates of the present invention, as depicted in formulas (II)and (II'), may be synthesized by the solution phase method shown in FIG.3. This solution phase method of preparation of the intermediates ispreferred.

Utility and Formulation

As discussed in the Background section, factor Xa catalyzes theformation of thrombin which is the penultimate reaction in thecoagulation cascade common to both the intrinsic and extrinsicinitiation pathways which terminate in the formation of a fibrin clot.Inhibitors of factor Xa would therefore inhibit fibrin deposition,thrombus formation and the consumption of coagulation proteins.Accordingly, the compounds of the present invention are thought to beuseful either as in vitro diagnostic reagents for selectively inhibitingin a sample factor Xa without inhibiting factor XIa, thrombin or tissueplasminogen activator (tPA), or as pharmacological agents for preventingor treating certain thrombotic disorders.

The compounds of the present invention are distinquished by theirspecificity for factor Xa, that is, their ability to inhibit thecatalytic activity of factor Xa while not appreciably inhibiting thecatalytic activity of factor XIa, thrombin and tPA. This specificity ofthe described inhibitors of actor Xa is an important feature of thecompounds of the present invention with respect to their ability toinhibit thrombus formation. The importance of specifically inhibitingfactor Xa versus thrombin as demonstrated by the compounds embodied inthis application may be better understood if one considers the amplifiednature of the coagulation cascade where one molecule of factor Xa canresult in the generation of 200,000 thrombin molecules per minute.Therefore, the amount of a selective factor Xa inhibitor required toachieve a relevant in vitro or in vivo antithrombotic effect will beconsiderably less than a comparable thrombin inhibitor of equal potencyor another inhibitor of thrombus formation which lacks this specificity.

The use of stoppered test tubes having vaccum therein as a means to drawblood obtained by venepuncture into the tube is well known in themedical arts. Kasten, B. L., "Specimen Collection", Laboratory TestHandbook, 2nd Edition, Lexi-Comp Inc, Cleveland pp. 16-17 (Edits.Jacobs, D. S. et al. 1990). Such vacuum tubes may be free ofclot-inhibiting additives, in which case, they would be useful for theisolation of mammalian serum from the blood. They may alternativelycontain clot-inhibiting additives (such as heparin salts, EDTA salts,citrate salts or oxalate salt), in which case, they would be useful forthe isolation of mammalian plasma from the blood. The compounds of thepresent invention are thought useful as additives for incorporation intoblood collection tubes to prevent clotting of the blood drawn into them.As such, the compounds of the present invention would be useful as invitro diagnostic reagents.

Inhibitors of factor Xa would be useful pharmacological agents for thetreatment of many thrombotic disorders including, myocardial infarction,unstable angina, disseminated intravascular coagulate on and associatedcomplications resulting from venous thrombosis. In addition, theseinhibitors would be useful as adjunctive or conjunctive agents toprevent recurrent thrombosis following enzymatic thrombolysis andpercutaneous transluminal angioplasty. Furthermore, specific inhibitorsof factor Xa may be useful in the suppression of metastatic migration ofcertain tumor types as described by Tuszynski, G. P. et. al., "Isolationand characterization of antistasin, an inhibitor of metastasis andcoagulation", J. Biol. Chem, 262:9718-9723 (1987) and Brankamp, R. G.et. al., "Ghilantens: anticoagulants, antimetastatic proteins from theSouth American leech Haementeria ghilianii"", J. Lab Clin. Med.,115:89-97 (1990).

The specificity of the described inhibitors of factor Xa is an importantfeature of the compounds of the present invention with respect to theirability to control pathogenic thrombosis formation with minimal effectsof the hemostatic potential of the treated patient. This will result ina reduction in the incidence of associated bleeding complications duringtherapy. The specificity of the described factor Xa inhibitors versustPA is absolutely required if these compounds are to be usedconjunctively with this thrombolytic agent in the reperfusion ofinfarct-related coronary vessels. Overall, the more specificity theinhibitor exhibits towards individual enzymes in the coagulationcascade, the less probability exists that unwanted side effects willoccur during therapy. Accordingly, the compounds of the presentinvention are thought useful as pharmacological agents for preventing ortreating certain in vivo thrombotic disorders.

To assay their activities, the compounds of the present invention aredissolved in buffer to give solutions containing concentrations rangingunder assay concentrations from 0 to about 100 μM. The enzyme to betested is then added to a solution containing a specified concentrationof the test compound. Then after an incubation period, syntheticsubstrate for the enzyme being tested is added. The rate of substrateturnover is determined spectrophotometrically. The IC₅₀ of the testcompound is determined for each test compound in assays for factor Xa,factor XIa, thrombin and tPA. IC₅₀ is that concentration of testcompound giving 50% inhibition of the substrate turnover. Percentselectivity is used to indicate selectivity of a compound in inhibitingFactor Xa in comparison with either Factor XIa, thrombin or tPA. Percentselectivity of a particular compound for either Factor XIa, thrombin ortPA refers to a number obtained by dividing the product of one hundredand the IC₅₀ of the compound for Factor Xa by the IC₅₀ of the compoundfor either Factor XIa, thrombin or tPA.

Preferred are those compounds of formulas I and I' for which the percentSelectivity for factor XIa, thrombin and tPA are each less than or equalto 10. The Percent Selectivity of each inhibitor for factor Xa is takenas 100. A Percent Selectivity less than 100 for a given compoundindicates it is a stronger inhibitor of Factor Xa than of either ofFactor XIa, thrombin or tPA. The smaller the percent selectivity, theless active that compound is for inhibition of factor Xia, thrombin ortPA.

The present invention provides compounds, their pharmaceuticallyacceptable salts and pharmaceutically acceptable compositions preparedfrom them which are thought useful as potent and specific inhibitors offactor Xa, both in vitro and in vivo. In mammals, the in vivo uses wouldinclude their administration as a therapeutic agent to prevent theformation of fibrin clots in blood vessels resulting from the presenceof factor Xa, to prevent abnormal thrombus formation resulting fromthrombotic disorders, and to prevent or treat the recurrent thrombusformation resulting from chemical or mechanical intervention directed toclearing blocked vessels. Additionally, the compounds, their salts andvarious compositions derived therefrom are thought useful as therapeuticagents for suppressing the metasiatic migration of tumor types inmammals by virture of their inhibitory properties.

The present invention also encompasses the pharmaceutically acceptablesalts of the compounds of formulas (I) and (I'). These salts includeacid addition salts, for example, salts of hydrochloric acid,hydrobromic acid, acetic acid, benzene sulfonic acid and other suitableacid addition salts.

The present invention also encompasses compositions prepared for storageand subsequent administration which comprise a therapeutically effectiveamount of a compound of the present invention in a pharmaceuticallyacceptable carrier or diluent. Pharmaceutically acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. For example, sodium benzoate, sorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Id.at 1449. In addition, antioxidants and suspending agents may be used.Id.

The compositions of the present invention may be formulated and used astablets, capsules or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxilliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g, liposomes) may be utilized.

The present invention also includes a method for preventing or treatinga condition in mammals characterized by abnormal thrombosis. Thetherapeutically effective amount of the composition required as a dosewill depend on the route of administration, the type of mammal beingtreated, and the physical characteristics of the specific mammal underconsideration. The dose can be tailored to achieve optimal efficacy butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.

In practicing the methods of the invention, the compounds orcompositions can be used alone or in combination with one another, or incombination with other therapeutic or diagnostic agents. These compoundscan be utilized in vivo, ordinarily in a mammal, preferably in a human,or in vitro. In employing them in vivo, the compounds or compositionscan be administered to the mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, nasally or intraperitoneally, employing a varietyof dosage forms.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, willbe within the ambit of one skilled in the art. Typically, applicationsof compound are commenced at lower dosage levels, with dosage levelbeing increased until the desired effect is achieved.

The dosage for the compounds of the present invention can range broadlydepending upon the desired affects and the therapeutic indication.Typically, dosages will be between about 0.01 μg/kg and 100 mg/kg bodyweight, preferably between about 0.01 μg/kg and 10 mg/kg body weight.Administration is preferably parenteral, such as intravenous on a dailybasis.

To assist in understanding the present invention, the following examplesare included which describe the results of a series of experiments. Thefollowing examples relating to this invention should not, of course, beconstrued as specifically limiting the invention. Variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

The invention will now be further illustrated by the following examples.The Examples 1 to 7 illustrate the reaction scheme of FIG. 1. Examples18 through 20 illustrate the reaction scheme of FIG. 2. Examples 14through 17, 21 and 22 illustrate the reaction scheme of FIG. 3.

EXAMPLES Example 1

Preparation of α-N-t-butoxycarbonyl-N^(g) -nitroargininal ##STR11##

The following procedure for the synthesis of α-N-t-butoxy-carbonyl-N^(g)-nitro-argininal (1) is a modification of the procedure of Fehrentz, J.A. and Castro, B, Synthesis, 676 (1983).

Boc-N^(g) -nitroarginine was obtained from Calbiochem. N-methylpiperidine, N,O-dimethylhydroxyl amine hydrochloride andisobutylchloroformate, and lithium aluminum hydride may be obtained fromAldrich Chemical Company, Inc. Dichloromethane, ethyl acetate, methanoland tetrahydrofuran may be obtained from Fisher scientific Company.

N-methyl piperidine (11.4 mL, 90 mmole) was slowly added to a stirredsuspension of N,O-dimethylhydroxyamine (8.42 g, 94 mmole) in 75 mLdichloromethane which had been cooled to about 0° C. The solution wasallowed to stir for 20 minutes then was kept cold for use in the nextstep. In a separate flask, Boc-N^(g) -nitroarginine (30.0 g, 94 mmole)was dissolved by heating in about 1400 mL of tetrahydrofuran and cooledunder nitrogen to 0° C. A mixture of N-methyl piperidine (11.4 mL, 90mmole) and isobutyl chloroformate (12.14 mL, 94 mmole) was added and themixture stirred for 10 minutes. The free hydroxylamine prepared abovewas added all at once and the reaction mixture was allowed to warm toroom temperature then stirred overnight.

The resulting precipitate was removed by filtration then washed with 200mL of tetrahydrofuran. After concentrating the filtrates to about 150 mLunder vacuum, 400 mL of ethyl acetate was added, followed by ice to coolthe solution. The cooled solution was washed with two 75 mL portions of0.2N hydrochloric acid, two 75 mL portions of 0.5N sodium hydroxide, oneportion of 75 mL of brine, then dried over anhydrous magnesium sulfate.Upon concentration in vacuum, 22.7 g (70% yield) of solid Boc-N^(g)-nitroarginine N-methyl-O-methylcarboxamide was recovered. Thin layerchromatographic analysis in 9:1 dichloromethane/methanol(silica gel)showed one spot.

A flask was placed under a nitrogen atmosphere charged with 70 mL of 1Nlithium aluminum hydride in tetrahydrofuran and 500 mL of drytetrahydrofuran then cooled to -50° C. A solution containing Boc-N^(g)-nitroarginine N-methyl-O-methylcarboxamide (23 g, 66 mmole) in 50 mLdry tetrahydrofuran was slowly added while the temperature of thereaction mixture was maintained at -50° C. After allowing the reactionmixture to warm to 0°C. by removal of the cooling, it was recooled to-30°C., at which temperature, 100 mL (0.2 mole) of 2N potassiumbisulfate was added with stirring over a 15 minute period. The reactionmixture was then allowed to stir at room temperature for 30 minutes. Theresulting mixture was filtered and the filtrate was concentrated to 100mL under vacuum. The concentrate was diluted with 800 mL ethyl acetate,then was washed with two 50 mL portions of 1N hydrochloric acid, two 50mL portions of saturated sodium bicarbonate, and one 50 mL portion ofbrine. The combined aqueous extracts were extracted with three 100 mLportions of ethyl acetate. All of the ethyl acetate washes werecombined, then dried over anhydrous magnesium sulfate. The mixture wasconcentrated under vacuum to yield 13.6 g (70%) of the titled compound.

Example 2

Preparation of trans-4-(aminomethyl)-cyclohexane carboxylic acid benzylester para-touluenesulfonate salt ##STR12##

Trans-4-(aminomethyl)-cyclohexane carboxylic acid (50 g, 0.318 moles),p-toluenesulfonic acid monhydrate (61.7 g, 0.324 moles), benzyl alcohol(250 mL, 2.4 moles) and 250 mL of toluene were combined and stirred. Themixture was refluxed for 24 hours and the liberated water was removed bymeans of a Dean-Stark apparatus. A clear solution was obtained after 5hours of refluxing. The solution was allowed to cool to room temperaturewhereupon the product crystallized. The mixture was vacuum filtered,washed with ether and dried in a vacuum oven to give 128.12 g (96%yield). Greenstein, J. P. and Winitz, M, "Chemistry of the Amino Acids",Robert E. Krieger Publishing Company, Malabar, Florida, Vol. 2, p942(1986). ¹ H NMR (CD₃ OD) δ 1.05 (m, 2H), 1.43 (m, 2H), 1.59 (m, 1H),1.85 (m, 2H), 2.03 (m, 2H), 2.33 (m, 1H), 2.35 (s, 3H), 2.75 (d, 2H),5.09 (s, 2H), 7.23 (d, 2H), 7.32 (m, 5H), 7.69 (d, 2H). M.P. 154°-156°C.

Example 3

Preparation of 1-t-butoxycarbonyl-semicarbazidyl-trans-4-methylcyclohexane carboxylic acid benzyl ester ##STR13##

Carbonyldiimidazole (3.24 g, 0.02 moles) was dissolved in 45 mL ofdimethylformamide (DMF) at room temperature under nitrogen. A solutionof t-bury carbazate (2.48 g, 0.02 moles) in 45 mL of DMF was addeddropwise. The solid benzyl ester 2 (8.38 g, 0.02 moles) was then added,followed by the dropwise addition of 3.06 mL of triethylamine over a 30minute period. The reaction was allowed to stir at room temperatureunder nitrogen for one hour. 100 mL of water was added and this mixturewas extracted three times with 50 mL of ethyl acetate. The ethyl acetatelayers were combined and extracted two times each with 75 mL of 1Nhydrochloric, water, saturated sodium bicarbonate, brine and dried withanhydrous magnesium sulfate. The mixture was filtered and the solutionwas concentrated to give an oil. This material could be purified byrecrystallization from ethyl acetate/hexanes (M.P.=106-108°C.) or useddirectly in the next step. ¹ H NMR (CDCl₃) δ 0.94 (m, 2H), 1.42 (m, 2H),1.45 (s, 9H), 1.81 (m, 2H), 2.02 (m, 2H), 2.27 (m, 1H), 3.17 (t, 2H),509 (s, 2H), 5.51 (t, 1H), 6.46 (s, 2H), 7.34 (m, 4H).

Example 4

Preparation of1-(t-butoxycarbonyl)-3-semicarbazidyl-trans-4-methyl-cyclohexanecarboxylic acid ##STR14##

The crude Boc-benzyl ester 3 from above in 250 mL of methanol wascombined with 500 mg of 10% palladium on activated carbon. After shakingon the hydrogenator for one hour at 5 psig, the mixture was filteredthrough a pad of diatomaceous earth in a fine filtered filter. Thefiltrate was concentrated to a foam, methylene chloride was added and aprecipitate formed. The crystallized material was filtered and washedwith ether. This yielded 4.0 g of crude product (12.7 mmoles; yield 62%overall yield from compound 2) ¹ H NMR (CD₃ OD), δ 0.96, (m, 2H), 1.42(m, 2H), 1.46 (s, 9H), 1.82 (m, 2H), 1.97 (m, 2H), 2.18 (m, 1H), 3.0 (t,2H). M.P.=185°-189° C.

Example 5

Preparation of semicarbazidyl-trans-4-methyl cyclohexane carboxylic acidtrifluoroacetate salt ##STR15##

Compound 4 (315 mg, 1 mmole) was added to 10 mL of trifluoroacetic acidat 0°C. and the resulting solution was allowed to stir for 30 minutes.After this time the solution was added dropwise to 75 mL of dry ether. Aprecipitate formed, and the mixture was filtered and washed with ether.Weight of crude product was 254 mg (77% yield). ¹ H NMR (CD₃ OD), δ 1.0(m, 2H), 1.38 (m, 2H), 1.43 (m, 1H), 1.84 (m, 2H), 2.01 (m, 2H), 2.22(m, 1H), 3.04 (d, 2H). M.P.=154°-156° C.

Example 6

Preparation of α-(t-butoxycarbonyl)-N^(g) -nitroargininal-semicarbazonyl-trans-4-methyl-cyclohexane carboxylic acid##STR16##

A solution of 5 (13.7 g, 41.6 mmoles) and crude 1 (18.0 g, 59 mmoles) in135 mL of ethanol, containing 45 mL of water, was treated with sodiumacetate trihydrate (9.41 g, 69 mmole) and refluxed for one hour. Thissolution was allowed to cool and then poured into 0.1N hydrochloric acidand extracted with three times using 100 mL of ethyl acetate perextraction. The combined organic phases were washed with water, brine,dried over anhydrous magnesium sulfate and concentrated to a smallvolume. This cloudy mixture was allowed to set overnight at 5° C. toprecipitate the product, which was isolated by filtration and driedunder vacuum. This gave 9.9 g, 47% yield based on 5. ¹ H NMR (CD₃ OD),δ1.0 (m, 2H), 1.43 (s, 9H), 1.45-2.20 (m, 13H), 3.09 (d, 2H), 3.30 (m,2H), 4.18 (bs, 1H), 7.10 (d, 1H). M.P.=162°-163° C.

Example 7

Synthesis of Semicarbazone Solid Support ##STR17##

Solid phase reagent 7 was prepared by placing methyl-benzhydralamine(MBHA) (0.8 g, 0.5 mmoles, 0.62 g/mole) resin in a reaction vessel andwashing one time with dichloromethane (DCM) (all washes require 10 mL ofsolvent with agitation for 1 to 2 minutes), three times with dimethylformamide (DMF), two times with 10% diisopropylethyl amine (DIEA)/DMF,and four times with DMF. 5 mL of DMF, 4-methylmorpholine (102 μL, 1mmole),benzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate(BOP reagent, 443 mg, 1 mmole) and compound 6 (500 mg, 1 mmole) wereadded, mixed on a rotating wheel for 16 hours, and washed three timeswith DMF, two times with 10% DIEA/DMF and three times with DMF. Theresin was then washed successively with DCM, methanol and ether. Theresulting resin 7 showed a 98-99% coupling yield by ninhydrin.

This resin was then extended at the N-terminus, with amino acids oramino acid analogs, on a conventional peptide synthesizer using standardt-Boc methodology as shown in the examples which follow.

The synthesis of the peptide analogs was performed on an AppliedBiosystems Model 430A peptide synthesizer using the t-Boc chemistryconditions in the 430A user's manual. The resulting protected peptidealdehyde can be cleaved from the support with a mixture of aqueous acidand formaldehyde, and then deprotected with hydrogen/Pd. The nitro groupcan be removed from the guanidine group without reduction of thealdehyde.

Example 8

Preparation ofN-t-butoxycarbonyl-D-3-(2-naphthyl)alanyl-L-phenylalanyl-L-argininal##STR18##

The above peptide aldehyde was synthesized using an Applied BiosystemsModel 430A peptide synthesizer. The Boc chemistry conditions utilizedwere as provided in the instrument user's manual.

Resin 7 (1.00 g, 0.500 mmole) was made ready for use by removing the Bocprotecting groups by treatment with 50% trifluoroacetic acid (indichloromethane). After washing and neutralizing the acidity bytreatment with 10% diisopropyl ethylamine (in dichloromethane),commercially available Boc-protected amino acids were coupled to thesupport reagent (and the growing amino acid support chain) in asequential manner.

Thus, N-Boc-L-phenylalanine was attached to the resin usingdicyclohexylcarbodiimide and 1-hydroxybenztriazole in dimethylformamide,followed by treatment with 50% trifluoroacetic acid (in dichloromethane)to remove the Boc protecting group, a wash step and a wash with 10%diisopropylethylamine (in dichloromethane) to neutralize acidity.N-Boc-D-3-(2-naphthyl )alanine was coupled in the same manner. Thetreatment with 50% trifluoroacetic acid was omitted after the lastcoupling.

The peptide aldehyde was removed from the solid phase, by treatment witha mixture comprising 5 mL of tetrahydrofuran, 1 mL of acetic acid, 1 mLof formaldehyde and 0.100 mL of 1N hydrochloric acid for 1 hour withstirring. After filtering this mixture, the resin was washed with 10 mLof tetrahydrofuran. The combined filtrates were diluted with 100 mLwater and extracted with ethyl acetate. The ethyl acetate phase was thenwashed with saturated sodium chloride, dried over magnesium sulfate, andconcentrated under vacuum.

To remove the nitro and benzyl (where applicable) protecting groups ofthe peptide aldehyde, the concentrated peptide aldehyde was taken up ina mixture 10 mL of 10% water in methanol, 0.300 mL of 1N hydrochloricacid and 0.200 g of palladium on carbon, then treated with hydrogen at 5psig for 45 minutes. The mixture was filtered through a fine frittedfilter with diatomaceous earth, washed with 10% water in methanol andconcentrated to give the crude peptide aldehyde.

The resulting peptide aldehyde is then purified using reverse phase HPLCon a 10 micron particle size, 300 angstrom pore size C-18 column,eluting with a water-acetonitrile (both containing 0.1% trifluoroaceticacid) gradient, where the gradient ran from 5% to 40% acetonitrile. Thecolumn fractions were analyzed by analytical HPLC and fractionscontaining pure product were pooled and lyophilized to yield theabove-identified product. Fast atom bombardment mass spectrometry gaveobserved molecular weight of 602.5 a.m.u.; calculated molecular weightwas 602.3 a.m.u.

Example 9

Preparation ofN-t-Butoxycarbonyl-D-2-Phenylglycyl-L-phenylalanyl-L-Arainial ##STR19##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 8. Here, N-Boc-L-phenylalanine was firstattached to resin 7 followed by N-Boc-D-phenylglycine. As in Example 8,the treatment with 50% trifluoroacetic acid was omitted after the lastcoupling step. Fast atom bombardment mass spectrometry gave an observedmolecular weight of 538.3 a.m.u; calculated molecular weight was 538.3a.m.u.

Example 10

Preparation ofN-t-butoxycarbonyl-D-phenylalanyl-L-3-(2-naphthyl)alanyl-L-argininal##STR20##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 8. Here, N-Boc-L-3-(2-naphthyl)alaninewas first attached to resin 7, followed by N-Boc-D-phenylalanine. As inExample 8, the treatment with 50% trifluoroacetic acid was omitted afterthe last coupling step. Fast atom bombardment mass spectrometry gave anobserved molecular weight of 602.3 a.m.u; calculated molecular weightwas 602.3 a.m.u.

Example 11

Preparation ofN-t-butoxycarbonyl-D-phenylalinyl-L-phenylalinyl-L-argininal ##STR21##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 8. Here, N-Boc-L-phenylalanine was firstattached to resin 7 followed by N-Boc-D-phenylalanine. As in Example 8,the treatment with 50% trifluoroacetic acid was omitted after the lastcoupling step. Fast atom bombardment mass spectrometry gave an observedmolecular weight of 552.5 a.m.u; calculated molecular weight was 552.6a.m.u.

Example 12

Preparation ofN-t-butoxycarbonyl-D-phenylalanyl-L-3-(1-naphthyl)alanyl-L-argininal##STR22##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 8. Here, N-Boc-L-3-(1-naphthyl)alaninewas first attached to resin 7 followed by N-Boc-D-phenylalanine. As inExample 8, the treatment with 50% trifluoroacetic acid was omitted afterthe last coupling step. Fast atom bombardment mass spectrometry gave anobserved molecular weight of 602.4 a.m.u; calculated molecular weightwas 602.7 a.m.u.

Example 13

Preparation ofN-acetyl-D-phenylalanyl-L-3-(1-naphthyl)alanyl-L-argininal ##STR23##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 8. Here, N-Boc-L-3-(1-naphthyl)alaninewas first attached to resin 7 followed by N-acetyl-β-phenylalanine. Asin Example 8, the treatment with 50% trifluoroacetic acid was omittedafter the last coupling step. Fast atom bombardment mass spectrometrygave an observed molecular weight of 544.3 a.m.u; calculated molecularweight was 544.3 a.m.u.

Example 14

Preparation of 1-t-butoxycarbonyl-semicarbazidyl-4-diphenylmethane.##STR24##

A solution of carbonyldiimidazole (16.2 g, 0.10 mole) indimethylformamide (DMF, 225 mL) was prepared at room temperature andallowed to stir under nitrogen. A solution of t-butyl carbazate (13.2 g,0.10 mole) in DMF (225 mL) was then added dropwise over a 30 minuteperiod. Next, diphenylmethylamine (18.3 g, 0.10 moles) in DMF (100 mL)was added over a 30 minute period. The reaction was allowed to stir atroom temperature under nitrogen for one hour. Water (10 mL) was addedand this mixture was concentrated to about 150 mL under vacuum. Thissolution was poured into water (500mL) and extracted with ethyl acetate(400 mL). The ethyl acetate phase was extracted two times each with 75mL 1N hydrochloric acid, water, saturated sodium bicarbonate, brine anddried with anhydrous magnesium sulfate. The mixture was filtered and thesolution was concentrated to give 29.5 g (85% yield) of a white foam.This material could be purified by recrystallization from ethylacetate/hexane, but was pure enough to use directly in the next step:M.P.=142°-143° C. Anal. Calcd. for C₁₉ H₂₃ N.sub. O₃ : C, 66.84; H,6.79; N, 12.31. Found: C, 66.46; H, 6.75; N; 12.90.

Example 15

Preparation of semicarbazidyl-4-diphenylmethane trifluoroacetate salt##STR25##

A solution of compound 24! (3.43 g, 10 mmole) in dichlormethane (12.5mL) and trifluoroacetic acid (12.5 mL) was stirred at room temperaturefor 30 minutes. After this time the solution was added dropwise to ether(75 mL). A precipitate formed, and the mixture was filtered and washedwith ether. Weight of crude product was 2.7 g (80% yield): mp 182°-184°C.

Example 16

Preparation of α-N-(t-butoxycarbonyl)-N^(g)-nitro-argininal-semicarbazonyl-4-N-diphenylmethane ##STR26##

A solution of compound 25! (2.65 g, 7.8 mmoles) and 1(α-N-(t-butoxycarbonyl)-N^(g) -nitro-argininal, 2.36 g, 7.8 mmoles) inethanol (20 mL) containing water (20 mL) was treated with sodium acetatetrihydrate (1.2 g, 8.8 mmoles) and refluxed for one hour. This solutionwas allowed to cool and then poured into water and extracted three timeswith ethyl acetate. The combined organic phase was washed with water,0.1N hydrochloric acid, brine, dried with anhydrous magnesium sulfate,and concentrated to a small volume. The white solid residue wasrecrystallized from acetonitrile/ether. This gave 3.2 g (78% yield basedon compound of Example 1: M.P.=78°-79° C.

Example 17

Preparation of N^(g) -nitro-argininal-semicarbazonyl-4-N-diphenylmethanetrifluoroacetate salt ##STR27##

A solution of compound 26! (1.2 g, 8.8 mmoles) in dichloromethane (5 mL)and trifluoroacetic acid (5 mL) at room temperature was allowed to stirfor 30 minutes. After this time, the solution was added dropwise toether (40 mL). A precipitate formed, and the mixture was filtered andwashed with ether. This gave 0.51 g of a pure white solid salt (97%yield): M.P.=159°-160° C.

Example 18

Preparation of L-3-(1-naphthyl)alanine-O-benzyl ester p-toluenesulfonicacid salt ##STR28##

L-3-(1-naphthyl)alanine (25.0 g, 115 mmole), p-toluenesulfonic acidmonohydrate (24.1 g, 127 mmole), toluene (1250 mL) and benzyl alcohol(29.8 mL, 288 mmole) were combined and refluxed with stirring overnight.Water (4.1 mL) was removed by a Dean-Stark trap. After cooling to roomtemperature, the resulting suspension was poured into 1000 mL of etherand stirred for 10 minutes. The solid was then filtered, washed with1000 mL of ether and dried under vaccum to give 50.5 g (92% yield) ofthe title compound. M.P.=161°-163° C.

Example 19

Preparation ofα-N-(t-butoxycarbonyl)-D-phenylalanyl-L-3-(1-naphthyl)alanine-O-benzylester ##STR29##

A solution of compound 28! (48.5 g, 101 mmole), Boc-D-phenylalanine(26.7 g, 101 mmole) andbenzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate(BOP, 44.7 g, 101 mmole) was prepared in 240 mL of DMF. The reactionmixture was cooled to 0° C., then 33.3 mL of 4-methylmorpholine (NMM)was added with stirring. After stirring overnight, the reaction mixturewas added to 800 mL of water and then extracted with two 400 mL portionsof ethyl acetate. The organic layer was washed with an equal volume of1N citric acid, water, saturated sodium chloride, and then was driedover anhydrous magnesium sulfate. The organic layer was concentratedunder vacuum to give a solid. The solid was then recrystallized fromethyl. acetate/hexanes to give 42 g (76% yield) of the pure crystallinetitle compound. M.P.=151°-153° C.

Example 20

Preparation ofα-N-(t-butoxycarbonyl)-D-phenylalanyl-L-3-(1-naphthyl)alanine ##STR30##

A solution of compound 21! (38 g, 69 mmole) in 1000 mL oftetrahydrofuran was placed in a Parr apparatus. The reaction mixture waspurged of air with nitrogen gas for 30 minutes, then 38 g of 10%palladium on carbon which had been pre-moistened with 10 mL of water wasadded. After purging, the mixture was stirred for 2 hours at roomtemperature under 30 psig of hydrogen gas. After this time, the mixturewas filtered. The filtrate was concentrated to dryness under vacuum toyield 28 g (88% yield) of the title compound. M.P.=113°-115° C.

Example 21

Preparation ofα-N-(t-butoxycarbonyl)-D-phenylalanyl-L-3-(1-naphthyl)alanyl-L-Na-nitro-aragninal-semicarbazonyl-4-N-diphenylmethane ##STR31##

A solution of compound 27! (9.08 g, 16.7 mmole), compound 22! (7.72 g,16.7 mole), BOP (7.38 g. 16.7 mole), NMM (5.27 mL, 48 mole) and 50 mL ofDMF was prepared and cooled to 0° C. The reaction mixture was stirredfor 2 hours at this temperature. After this time, the reaction mixturewas poured into 500 mL of ethyl acetate. 150 mL of water was added andthis mixture was stirred for 20 minutes at room temperature. After thistime, the stirring was discontinued and the layers separated. Theorganic layer was separated, then washed with an equal volume ofsaturated citric acid, saturated sodium bicarbonate, water and saturatedsodium chloride, then was dried over anhydrous magnesium sulfate. Afterconcentrating the organic layer to dryness, the crude product wasredissolved in dichloromethane, then was chromatographed on a silica.gel column, eluting with 4 to 16% isopropyl alcohol (indichloromethane). The product eluted between 10 to 14% isopropyl alcohol(in dichloromethane). Fractions containing pure product were selected byuse of thin-layer chromatography on silica gel, developing with 10%methanol in dichloromethane and pooled. The pool. was reduced to drynessunder vacuum to give 7 g (46% yield) of the title compound.M.P.=140°-150° C. (decomposed).

Example 22

Preparation ofN-t-butoxycarbonyl-D-phenylalanyl-L-3-(1-naphthyl)alanyl-L-argininal##STR32##

A solution of compound 15! (1.95 g, 2.15 mmole) and 98 mL of methanolwas placed in a Parr vessel. 1M hydrochloric acid (3.9 mL), acetic acid(9.8 mL) and 10% palladium on carbon (1.95 g) pre-moistened with severaldrops of water were added, then the reaction mixture was purged of airwith nitrogen gas for 30 minutes. After purging, the mixture, it wasallowed to stir for 18 hours at room temperature under a 15 psigatmosphere of hydrogen gas. After this time, the palladium on carbon wasfiltered off and the filtrate was concentrated under vacuum to yield anoil.

The oil was redissolved in a solution comprised of 78 mL of methanol, 11mL acetic acid, 0.6 mL of 1M hydrochloric acid and 6 mL of 37%formaldehyde (by weight in water), then allowed to stir for 45 minutesat room temperature. After this time, 200 mL of water was added and thesolution was concentrated to about 200 mL under vacuum. 86 mL ofacetonitrile was added to the cloudy concentrate to yield a clarifiedsolution. The title compound was purified from the solution usingreverse phase preparative HPLC on a 10 micron particle size, 300angstrom pore size C-18 column, eluting with a water-acetonitrile (bothcontaining 0.1% trifluoroacetic acid) gradient. The gradient ran from30% to 75% acetonitrile-water (containing 0.1% trifluoroacetic acid).Fractions which contained the title compound eluted at 50-58%acetonitrile-water. The fractions were pooled and lyophilized to give0.89 g (69% yield). Fast atom bombardment mass spectrometry gaveobserved molecular weight of 602.3 a.m.u.; calculated molecular weightwas 602.3 a.m.u.

Example 23

Preparation of N-Boc-D-phenylalanyl-L-prolyl-L-argininal ##STR33##

The above peptide aldehyde 23! has been described as a potent inhibitorof thrombin. See, e.g., Bajusz, S. et al., Folia Haematol. Leipzig,109:16 (1982); Bajusz, S., Symposia Biologica Hungarica, 25:277 (1984);and Bajusz, S. et al., J. Med. Chem, 33:1729 (1990). Accordingly, it wassynthesized for use as a comparison compound in the assays described inExample A.

Peptide aldehyde 23! was synthesized and purified in the same manner asdescribed in Example 8. N-Boc-L-proline was first attached to resin 7followed by N-Boc-D-phenyl alanine. The treatment with 50%trifluoroacetic acid was omitted after the last coupling. The titlecompound was obtained after further deprotection and purification. Fastatom bombardment mass spectrometry gave observed molecular weight of 502a.m.u.; calculated molecular weight was 502 a.m.u.

Example 24

Preparation of N-Boc-L-(α-biphenyl)glycine ##STR34##

15 g (82 mmole) of 4-biphenylcarboxaldehyde, 3.4 g (63 mmole) ofammonium chloride, 16.4 g (205 mmole) ammonium carbonate and 6.15 g (94mmoles) of potassium cyanide were dissolved together in 50% ethanol (indeionized water), placed under an atmosphere of argon, then heated forabout 12 to 16 hours at 40° C. After this time, the solid which hadformed in the mixture was filtered off and washed successively with 25mL of 50% ethanol (in deionized water), deionized water and diethylether to yield 30 g of the hydantoin solid. This solid was dissolved inhot methanol and triturated with deionized water.

4 g of the solid was added to 80 mL of 1M sodium hydroxide and wasrefluxed for 12 to 16 hours. After this time, 80 mL of ethanol and 3.2 g(80 mmole) of solid sodium hydroxide was added, and the mixture wasfurther refluxed for 12 to 16 hours. The mixture was adjusted to pH 5with concentrated HCl to yield a solid. The solid was filtered off anddried for 12 to 16 hours under vacuum to yield 2.7 g (75% yield) of thetitle compound.

Example 25

Preparation ofN-t-butoxycarbonyl-D,L-(α-biphenyl)glycyl-L-3-(1-naphthyl)alanyl-L-argininal##STR35##

The above peptide aldehyde was synthesized using an Applied BiosystemsModel 430A peptide synthesizer. The Boc chemistry conditions utilizedwere as provided in the instrument user's manual.

Resin 7 (0.67 to 1.00 g, 0.500 mmole amino groups) was made ready foruse by removing the Boc protecting groups by treatment with 50%trifluoroacetic acid (in dichloromethane). After washing andneutralizing the acidity by treatment with 10% diisopropylethylamine (indichloromethane), commercially available Boc-protected amino acids werecoupled to the support reagent (and the growing amino acid supportchain) in a sequential manner.

Thus, N-Boc-L-3-(1-naphthyl)alanine (2.0 mmole in 2 mL ofN-methylmorpholine) was attached to the resin by coupling for one hourwith dicyclohexylcarbodiimide (2.0 mmole in 2 mL of N-methylmorpholine)and 1-hydroxybenztriazole (2.0 mmole in 3.3 mL of N-methyl morpholine),followed by treatment with 50% trifluoroacetic acid (in dichloromethane)to remove the Boc protecting group, a wash step and a wash with 10%diisopropylethylamine (in dichloromethane) to neutralize acidity.N-Boc-D,L-α-biphenylglycine was coupled in the same manner. Thetreatment with 50% trifluoroacetic acid was omitted after the lastcoupling.

The protected peptide aldehyde was removed from the solid phase, bytreatment with a mixture of 5 mL of tetrahydrofuran, 1 mL of aceticacid, 1 mL of formaldehyde and 0.100 mL of 1N hydrochloric acid for 1hour with stirring. After filtering this mixture, the resin was washedwith 10 mL of tetrahydrofuran. The combined filtrates were diluted with100 mL water and extracted with ethyl acetate. The ethyl acetate phasewas then washed with saturated sodium chloride, dried over magnesiumsulfate, and concentrated under vacuum.

To remove the nitro and benzyl (where applicable) protecting groups ofthe peptide aldehyde, the concentrated peptide aldehyde was taken up ina mixture 4.2 mL of methanol, 0.49 mL of 1N hydrochloric acid and 0.250g of palladium on carbon, then treated with hydrogen at 5 psi for 45minutes. The mixture was filtered through a fine fritted filter withdiatomaceous earth, washed with 10% water in methanol and concentratedto give the crude peptide aldehyde.

The resulting peptide aldehyde was then purified using reverse phaseHPLC on a 10 micron particle size, 300 angstrom pore size C-18 column(Vydac), eluting with a water-acetonitrile (both containing 0.1%trifluoroacetic acid) gradient, where the gradient ran from 20% to 50%acetonitrile. The column fractions were analyzed by analytical HPLC andfractions containing pure product were pooled and lyophilized to yieldthe above-identified product. Fast atom bombardment mass spectrometrygave observed molecular weight of 664.3 a.m.u.; calculated molecularweight was 664.3 a.m.u.

Example 26

Preparation of Mono-O-benzylsuccinic acid ##STR36##

10 g (100 mmole) of succinic anhydride, 10 mL (97 mmole) of benzylalcohol and 10 mL of triethylamine were combined, the mixture was heatedto reflux, allowed to reflux for about 5 minutes, and then was allowedto stir unheated for 1 hour. After this time, the mixture was pouredinto 250 mL of 1M hydrochloric acid and was extracted with 2-75 mLportions of ethyl acetate. The organic phases were combined, washed withdeionized water, washed with brine, and then were dried over anhydrousmagnesium sulfate. The organic phase was concentrated to oil which uponsitting yielded crystalline 19.6 g (94% yield) of the title compound.

Example 27

Preparation ofN-succinyl-D-phenylalanyl-L-3-(1-naphthyl)alanyl-L-argininal ##STR37##

The above peptide aldehyde was synthesized on the resin in the samemanner as described in Example 25. Here, N-Boc-L-3-(1-naphthyl)alaninewas first attached to resin 7, followed by N-Boc-D-phenylalanine,followed by mono-O-benzylsuccinic acid.

The titled product as a protected semicarbazone was removed from thesolid phase with the concurrent removal of the nitro protecting group,by treating the resin with a mixture of 0.8 mL of anisole and 12.0 mL ofhydrofluoric acid at -20° C. for 20 minutes. After removal of thehydrofluoric acid by evaporation at room temperature, the remainingsolid was extracted with 50 mL of 0.1M ammonium bicarbonate, followed by100 mL of deionized water. The extracts were combined and extractedtwice with diethyl ether. The aqueous layer was then frozen andlyophilized.

The semicarbazone protecting group was removed taking up the 1lyophilized solid in a solution of 5 mL of tetrahydrofuran, 1 mL aceticacid, 0.1 mL of 1M hydrochloric acid and 1 mL of 37% formaldehyde (byweight in water), then allowing the mixture to stir for 1 hour at roomtemperature. After this time, 20 mL of water was added and the solutionwas extracted with ethyl acetate. The ethyl acetate phase was thenwashed with saturated sodium chloride, dried over magnesium chloride,and concentrated to about 20 mL under vacuum. 8.6 mL of acetonitrile wasadded to the cloudy concentrate to yield a clarified solution. Theresulting peptide aldehyde is then purified using reverse phase HPLC ona 10 micron particle size, 300 angstrom pore size C-18 column (Vydac),eluting with a water-acetonitrile (both containing 0.1% trifluoroaceticacid) gradient, where the gradient ran from 20% to 50% acetonitrile. Thecolumn fractions were analyzed by analytical HPLC and fractionscontaining pure product were pooled and lyophilized to yield theabove-identified product. Fast atom bombardment mass spectrometry gaveobserved molecular weight of 602.3 a.m.u.; calculated molecular weightwas 602.3 a.m.u.

Example 28

Preparation ofN-(4-methylpentanyl)-D-phenylalanyl-L-(3-(1-naphthyl)alanyl-L-argininal##STR38##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 27. Here, N-Boc-L-3-(1-naphthyl)alaninewas first attached to resin 7, followed by N-Boc-D-phenyl alanine,followed by 4-methyl valeric acid. Fast atom bombardment massspectrometry gave observed molecular weight of 600.4 a.m.u.; calculatedmolecular weight was 600.3 a.m.u.

Example 29

N-t-butoxycarbonyl-D-phenylalanyl-L-(3-trans-phenyl)prolinyl-L-argininal##STR39##

The above peptide aldehyde was synthesized and purified in the samemanner as described in Example 25. Here, N-Boc-L-(3-trans-phenyl)prolinewas first attached to resin 7, followed by N-Boc-D-phenylalanine.

Example A

Specificity--Determination of IC₅₀

The specificity of the peptide aldehydes 1! through 10! was determinedby measurement of their IC₅₀ against factor Xa, factor XIa, thrombin andtissue plasminogen activator (tPA). Peptide aldehyde 23! which has beendescribed in the art was run as a comparison. A specific concentrationof enzyme and its substrate were challenged with varying concentrations:of inhibitor. IC₅₀ is that concentration of inhibitor giving 50%inhibition of catalytic activity, under the assay conditions Specificassay procedures used are presented below.

Table 1 shows the results of these assays for substrate specificity,wherein ">25" means less than 50% inhibition was observed at aninhibitor concentration of 25 μM. In this table, "β-NpAla" refers to3-(2-naphthyl)alanine also known as 3-(β-naphthyl )alanine; "PhGly"refers to 2-phenylglycine; and "α-NpAla" refers to 3-(1-naphthyl)alaninealso known as 3-(α-naphthyl)alanine.

                  TABLE 1                                                         ______________________________________                                        Table of IC.sub.50 s for Inhibitors.                                                          Com-                                                                          pound               (μM)                                   Inhibitor       Num-           IC.sub.50                                                                          Throm-                                    Compound        ber     Xa     XIa  bin   tPA                                 ______________________________________                                        Boc-D-(β-NpAla)-Phe-Arg-al                                                                1!     0.22   16   14    >25                                 Boc-D-PhGly-Phe-Arg-al                                                                         2!     0.64   13   >25   >25                                 Boc-D-Phe-β-NpAla-Arg-al                                                                  3!     0.89   25   25    >25                                 Boc-D-Phe-Phe-Arg-al                                                                           4!     0.21   25   >25   >25                                 Boc-D-Phe-(α-NpAla)-Arg-al                                                               5!     0.030  14   13    >25                                 Ac-D-Phe-(α-NpAla)-Arg-al                                                                6!     0.025  0.32 >25   >25                                 Boc-L-BPG1y-(α-NpAla)-                                                                   7!     2.5    >25  >25   >25                                 Arg-al                                                                        Succ-D-Phe-(α-NpAla)-Arg-al                                                              8!     0.023  20   >25   >25                                 4MeV-D-Phe-(α-NpAla)-Arg-al                                                              9!     0.17   >25  >25   --                                  Boc-D-Phe-(3-trans-PhPro)-                                                                     10!    0.625  >25  >25   >25                                 Arg-al                                                                        Boc-D-Phe-Pro-Arg-al                                                                           23!    5.7    1.8  0.025 1.1                                 ______________________________________                                    

Table 2 shows the Percent Selectivity for exemplar compounds of thepresent invention. Percent Selectivity is defined as the 100 times theIC₅₀ for factor Xa divided by the IC₅₀ of either factor XIa, thrombin ortPA. The Percent Selectivity of each inhibitor for factor Xa is taken as100. Accordingly, a Percent Selectivity of less than 100 for a giveninhibitor factor XIa, thrombin or tPA is indicative of weakly inhibitingactivity, if active at all to inhibit those enzymes while stronglyinhibiting factor Xa.

                  TABLE 2                                                         ______________________________________                                        Table of Percent Selectivity for Inhibitors.                                                  Com-                Selec-                                                    pound          Per- tivity                                    Inhibitor       Num-           cent Throm-                                    Compound        ber     Xa     XIa  bin   tPA                                 ______________________________________                                        Boc-D-(β-NpAla)-Phe-Arg-al                                                                1!     100    1.4  1.6   <0.9                                Boc-D-PhGly-Phe-Arg-al                                                                         2!     100    4.9  <2.6  <2.6                                Boc-D-Phe-(β-NpAla)-Arg-al                                                                3!     100    3.6  3.6   <3.6                                Boc-D-Phe-Phe-Arg-al                                                                           4!     100    0.8  <0.8  <0.8                                Boc-D-Phe-(α-NpAla)-Arg-al                                                               5!     100    0.2  0.2   <0.1                                Ac-D-Phe-(α-NpAla)-Arg-al                                                                6!     100    7.8  <0.1  <0.1                                Boc-D,L-BPGly-(α-NpAla)-                                                                 7!     100    <10  <10   <10                                 Arg-al                                                                        Succ-D-Phe-(α-NpAla)-Arg-al                                                              8!     100    0.12 <0.1  <0.1                                Mev-D-Phe-(α-NpAla)-Arg-al                                                               9!     100    <0.68                                                                              <0.68 --                                  Boc-D-Phe-(3-trans-PhPro)-                                                                     10!    100    <2.5 <2.5  <2.5                                Arg-al                                                                        Boc-D-Phe-Pro-Arg-al                                                                           23!    100    317  22,800                                                                              518                                 ______________________________________                                    

(a) Factor Xa Assay.

Enzyme activity was determined using as substrate, S2765(N-a-benzyloxycarbonyl-D-argininyl-L-glycyl-L-arginine-p-nitroanilidedihydrochloride) which was obtained from Kabi Diagnostica. The substratewas made up in deionized water prior to use.

Human factor Xa was obtained from Enzyme Research Laboratories. Theenzyme was diluted into TBSA prior to use.

The assay was run by combining in appropriate wells 50 μL of TBSA, 50 μLof inhibitor in TBSA or TBSA (as negative control) and 50 μL of 2 nMhuman Factor Xa or TBSA (as background control). After incubating thismixture for 60 minutes at room temperature, 50 μL of 1 mM S-2765 wasadded to each well and the initial rate of the change of the opticaldensity at 405 nm (OD₄₀₅ nm) for each well was determined. OD₄₀₅ nm wasmeasured every 10 seconds for 5 minutes.

(b) Factor XIa Assay.

Enzyme activity was determined using as substrate, S2366(L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide hydrochloride) whichwas obtained from Kabi Diagnostica. The substrate was made up indeionized water prior to use.

Human factor XIa was obtained from Enzyme Research Laboratories, Inc.The enzyme was diluted into TBSA prior to use.

The assay was run by combining in appropriate wells 50 μL of TBSA, 50 μLof inhibitor in TBSA or TBSA (as negative control) and 50 μL of 2 nMhuman Factor Xia or TBSA (as background control). After incubating thismixture for 60 minutes at room temperature, 50 μL of 6 mM S-2366 wasadded to each well and the initial rate of the change of the opticaldensity at 405 nm (OD₄₀₅ n_(m)) for each well was determined. OD₄₀₅ nmwas measured every 10 seconds for 5 minutes.

(c) Thrombin Assay.

Enzyme activity was determined using as substrate, S2238(D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride)which was obtained from Kabi Diagnostica. The substrate was made up indeionized water prior to use.

Human α-thrombin was obtained from Enzyme Research Laboratories, Inc.The enzyme was diluted into TBSA prior to use.

The assay was run by combining in appropriate wells 50 μL of TBSA, 50 μLof inhibitor in TBSA or TBSA (as negative control) and 50 μL of 4 nMhuman α-thrombin or TBSA (as background control). After incubating thismixture for 60 minutes at room temperature, 50 μL of 0.24 mM S-2238 wasadded to each well and the initial rate of the change of optical densityat 405 nm (OD₄₀₅ nm) for each well was determined. OD₄₀₅ nm was measuredevery 10 seconds for 5 minutes.

(d) tPA Assay.

Enzyme activity was determined using as substrate, Pefachrome tPA(O-methylsulfonate-D-hexahydrotyrosine-L-glycyl-L-argini ne-p-nitroanilide acetate salt) which was obtained from Centerchem, Inc. Thesubstrate was made up in deionized water prior to use.

Human recombinant t-PA (Activase®) was obtained. from Genentech, Inc.The enzyme was reconstituted with water, then diluted into TBSA prior touse.

The assay was run by combining in appropriate wells 50 μL of TBSA, 50 μLof inhibitor in TBSA or TBSA (as negative control) and 50 μL of 4 nMhuman recombinant tPA or TBSA (as background control). After incubatingthis mixture for 60 minutes at room temperature, 50 μL of 4 mMPefachrome tPA was added to each well and the initial rate of the changeof optical density at 405 nm (OD₄₀₅ nm) for each well was determined.OD₄₀₅ nm was measured every 10 seconds for 5 minutes.

We claim:
 1. A compound of the formula: ##STR40## wherein Ar has theformula: ##STR41## wherein X is independently selected from the groupconsisting of hydrogen, methyl, halogen, and ethyl;R₁ is selected fromthe group consisting of --(CH₂)₃ --NH--C (═NNO₂)--NH₂, andalkyl-substituted derivatives thereof, wherein each alkyl isindependently selected and has about 1 to about 7 carbon atoms; R₂ isselected from the group consisting of arylkyls of about 7 to about 15carbon atoms optionally substituted with 1 to 2 independently selectedalkyl groups of about 1 to about 4 carbon atoms; R₃ is selected from thegroup consisting of aryl of about 6 to about 14 carbon atoms optionallysubstituted with 1 to 2 independently selected alkyl groups of about 1to about 4 carbon atoms, aralkyl of about 7 to about 15 carbon atomsoptionally substituted with 1 to 2 independently selected alkyl groupsof about 1 to about 4 carbon atoms, and alkyl of about 1 to about 7carbon atoms; and R₄ is selected from the group consisting of alkyl ofabout 1 to about 12 carbon atoms, alkenyl of about 3 to about 6 carbonatoms, aryl of about 6 to about 14 carbon atoms, aralkyl of about 7 toabout 15 carbon atoms, alkoxy of about 1 to about 12 carbon atoms,alkenyloxy of about 3 to about 8 carbon atoms, aryloxy of about 6 toabout 14 carbon atoms, alkenyl of about 7 to about 15 carbon atoms andcarboxylalkyl of about 2 to about 7 carbon atoms; or pharmaceuticallyacceptable salts thereof.
 2. A compound according to claim 1, wherein R₁is --(CH₂)₃ --NH--C(═NNO₂)--NH₂.
 3. A compound of claim 2, wherein R₂ isselected from the group consisting of phenylmethyl, diphenyl methyl,biphenylmethyl and napthylmethyl, each optionally ring substituted with1 to 2 independently selected alkyl groups of about 1 to about 4 carbonatoms, and R₃ is selected from the group consisting of phenyl,phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl, napthyl, andnapthylmethyl, each optionally ring substituted with 1 to 2independently selected alkyl groups of about 1 to about 4 carbon atoms.4. A compound of claim 3, wherein R₂ is selected from the groupconsisting of phenylmethyl, 1-naphthyl methyl and 2-naphthylmethyl, andR₃ is selected from the group consisting of phenyl, phenylmethyl, and2-napthylmethyl.
 5. A compound of claim 4, wherein R₄ is selected fromthe group consisting of methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, buryl , pentyl, hexyl, cyclopentyl,cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, adamantyl ,adamantylmethyl, 2-propenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,5-hexenyl, 2-cyclopentenyl, phenyl, phenyl methyl, diphenylmethyl,biphenyl, biphenylmethyl, napthyl, naphthylmethyl, 1,1-dimethylethyloxy,2-methylpropyloxy, 2,2-dimethyl propyloxy, cyclopentyl,cyclopentylmethyloxy, cyclohexyloxy, cyclohexylmethyloxy, adamantyloxy,adamantylmethoxy, phenoxy, benzyloxy, biphenylmethyloxy, naptholoxy,napthylmethyloxy and 2-carboxyethyl.
 6. A compound of claim 5, whereinR₄ is 1,1-dimethylethyloxy.
 7. A compound of claim 5, wherein R₄ ismethyl.
 8. A compound of claims 5, wherein R₄ is 2-carbxyethyl.
 9. Acompound of claim 1 wherein R₄ is selected from the group consisting ofalkyl of 1 to about 12 carbon atoms, alkoxy of 1 to about 12 carbonatoms, and carboxyalkyl of 2 to about 7 carbon atoms.
 10. A compound ofclaim 1 selected from the group consisting of: ##STR42##